Construction of novel class I histocompatibility antigens by interspecies exon shuffling

Human and mouse class I histocompatibility antigens share considerable structural homology at both the protein and DNA sequence level. This homology has allowed the production of hybrid class I molecules by the reciprocal exchange of DNA sequences corresponding to equivalent domains of HLA-B7 and either H-2Ld or H-2Dd. It is shown that these genes give rise to protein products that are stably expressed on the surface of murine L cells after DNA-mediated gene transfer. These proteins express only those monoclonal antibody-defined H-2 determinants that are expected based on their genetic construction. The molecules have allowed the localization of a number of polymorphic and monomorphic HLA-specific epitopes. In all but one case, expression of an epitope on a domain does not appear to be influenced by the replacement of adjacent human domains with their murine equivalents, suggesting a considerable degree of structural independence of the domains. Cells expressing the hybrid molecules have also been tested as targets for a panel of HLA-B7-specific cytotoxic T cell clones. The results show that the polymorphic determinants recognized by these clones map to the alpha 1 and alpha 2 domains of the HLA-B7 molecule. No evidence for an influence of species-related amino acid sequence differences in the third extracellular domain on T cell recognition was seen. The results are discussed in light of the proposed domain structure of the class I proteins and the potential use of such molecules for further functional studies.     

Alloantigenic sites on class I major histocompatibility complex antigens: 61-69 region in the first domain of the H-2Kb molecule induces specific antibody and T cell responses

The most polymorphic residues in the first domain of class I major histocompatibility complex (MHC) molecules are in the 61-69 region. We have chosen the H-2Kb molecule for determining the role of this region in the induction of alloimmune responses. A synthetic peptide, Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly corresponding to this region was synthesized. T cells enriched from the lymph nodes of allostrain mice that were previously primed with H-2Kb containing cells or with the synthetic peptide in complete Freund's adjuvant undergo extensive in vitro proliferation in response to the synthetic (61-69)H-2Kb peptide. The response was dependent on the presentation of the (61-69)H-2Kb peptide by the syngeneic antigen-presenting cells and was blocked by anti-class II MHC monoclonal antibodies. This peptide fragment of class I MHC molecule activates only helper/inducer type T cells that are involved in the primary responses but not the effector cytotoxic T cells. When coupled to a carrier protein, (61-69)H-2Kb peptide induced antibodies in allostrain mice that bind to intact H-2Kb molecule. No antibodies or T cell responses could be induced in syngeneic H-2b mice. The antigenic site on the H-2Kb molecule recognized by two H-2Kb-specific monoclonal antibodies B8 X 3 X 24 and Y-25 was also mapped in the 61-69 region by direct binding to the synthetic peptide. Therefore the 61-69 region on the H-2Kb molecule represents the first defined sequence on a class I molecule that is directly involved in the induction of alloimmune responses.     

Secretion of a soluble class I molecule encoded by the Q10 gene of the C57BL/10 mouse

The DNA sequence of the Q10 genes appears to be highly conserved amongst strains of mice and has only been found to be transcribed in the liver. An examination of the nucleotide sequence of the exon that normally encodes the transmembrane domain of class I molecules suggested that the Q10 gene encodes a secreted protein. We have established this by showing that L cells transformed with an expression vector containing the Q10 gene secrete a class I molecule which was identified with an antiserum raised against a peptide predicted by the Q10 transmembrane exon. Both the L cell-derived Q10 molecule and a class I protein immunoprecipitated from serum with this anti-peptide antiserum have mol. wts. of approximately 38 000; the Q10 molecule secreted by L cells is heterogeneous in mol. wt. This heterogeneity was drastically reduced after endoglycosidase F treatment, suggesting that Q10 molecules secreted into the serum by the liver may be glycosylated differently from those secreted by L cells. Endoglycosidase F treatment of both the L cell and serum forms of the soluble molecule yielded two products with mol. wts. of approximately 32 000 and 35 000; this is consistent with the observation that the predicted Q10 protein sequence has two potential glycosylation sites. In contrast to previous published results, the Q10 molecule reacted with rabbit anti-H-2 antisera which is consistent with its greater than 80% homology to the classical transplantation antigens.     

Cytolytic T cells recognize a chimeric MHC class I antigen expressed in influenza A infected transgenic mice

A chimeric H-2Kd/Kk gene, called pC31, contains the extracellular alpha 1 domain of Kd origin whereas the rest of the molecule is of Kk origin. Disruption of the syngeneic alpha 1-alpha 2 structure results in a total abrogation of the function of the C31 protein as a restriction element for H-2Kd and Kk restricted T cells during virus infection. In an attempt to obtain information on the functional polymorphism of MHC class I antigens as restriction elements, we have introduced the pC31 gene into the germ line of C3H/He mice (H-2k). The pC31 gene was transcribed in all tissues examined and the expression pattern paralleled the endogenous H-2Kk gene. However, the mRNA for the transgene was approximately 10-times more abundant, which was reflected in an elevated expression of the C31 protein in transgenic splenocytes. Most of the C31 antigen was found intracellularly. The C31 antigen could condition transgenic cytotoxic T lymphocytes in a specific manner during influenza A virus infection and functioned as the restricting element during T cell lysis of the infected cells. These results suggest that entire exons may be exchanged between MHC class I genes and that this exchange can generate novel and functional restriction elements.     

Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: functional T cell recognition requires preservation of fine structural determinants

Studies of immune recognition of hybrid class I antigens expressed on transfected cells have revealed an apparent general requirement that the N(alpha 1) and C1(alpha 2) domains be derived from the same gene in order to preserve recognition by virus-specific H-2-restricted and allospecific T cells. One exception has been the hybrid DL antigen in which the N domain of H-2Ld has been replaced by that of H-2Dd. Cells bearing this molecule serve as targets for some virus and allospecific CTL. Because cells expressing the reciprocal hybrid LD (N domain of H-2Dd replaced by that of H-2Ld) antigen have not been available, it has not been possible to evaluate whether this exception stemmed from the relatedness of H-2Ld and H-2Dd or whether the DL antigen fortuitously preserved some function of the parent molecule as a rare exception. To assess this question, and to evaluate the contribution of the N and C1 domains of H-2Ld and H-2Dd to serologic and T cell recognition, we have constructed the reciprocal chimeric gene pLD (the N exon of H-2Ld substituted for that of H-2Dd), introduced this into mouse L cells by DNA-mediated gene transfer, and analyzed the expressed product biochemically, serologically, and functionally. Transformant L cells expressing either LD or DL antigens were both reactive with a number of anti-H-2Ld or anti-H-2Dd N/C1-specific monoclonal antibodies, indicating the preservation in the hybrid molecules of determinants controlled by discrete domains. Mab binding was generally greater with cells expressing hybrid DL antigen than with those transformants expressing LD molecules. Moreover, the amount of beta 2M associated with DL antigens was more than that associated with LD. Cells expressing hybrid DL antigens were recognized as targets by bulk and cloned allospecific anti-H-2Dd and anti-H-2Ld CTL, whereas cells expressing LD molecules were not recognized by any of the T cells tested. VSV-specific H-2Ld-restricted CTL failed to lyse VSV-infected targets expressing either DL or LD. These results indicate that T cell reactivity of cells expressing the DL hybrid antigen is an exception to the observed general requirement for class I antigens to possess matched N and C1 domains for functional T cell recognition by T cells restricted to parental antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of alpha 1 with alpha 2 region in class I MHC proteins contributes determinants recognized by antibodies and cytotoxic T cells

The structure-function relationship of individual coding regions of class I mouse major histocompatibility complex proteins was studied by a combination of recombinant DNA, gene transfer techniques, and serologic and functional characterization. To examine the role of alpha 1 and alpha 2 regions in antibody and CTL recognition, the third exon of H-2Dd, Kd, and Ld transplantation antigen genes was replaced by the homologous coding region of the Qa-2-coded class I gene, Q6. We have chosen to carry out the exon shuffling experiments between these two different types of class I genes, because they are structurally similar and did not evolve to carry out identical functions. Therefore, it is less likely that the hybrid proteins will fortuitously recreate alpha 1-alpha 2 controlled functionally important determinants. The replacement of H-2 alpha 2 coding region with its Q6 counterpart had different effects on the expression of the three genes. The mutant H-2Dd gene transfected into L cells was expressed at high levels and retained several of the serologic determinants found on parental H-2Dd and Q6 domains. The serologic epitopes on the mutant H-2Kd-transfected cells were detectable at very low levels, whereas the product of the mutant H-2Ld gene could not be identified at all. Analysis of cells transfected with mutant H-2Dd gene with alloreactive and minor antigen(s)-restricted cytotoxic T cells indicated that the hybrid proteins lost the ability to be recognized by T cells. Our data suggest that cytotoxic T cells recognize conformational determinants composed of amino acids from alpha 1 and alpha 2 regions. Alternatively, it could be proposed that T cell recognition sites located in a single alpha 1 or alpha 2 protein region are susceptible to distortion upon alpha 1-alpha 2 interactions. Such susceptibility to conformational changes of the amino-terminal domain of transplantation antigens could be of functional importance for H-2-restricted antigen presentation.     

Endocytosis of the class I major histocompatibility antigen via a phorbol myristate acetate-inducible pathway is a cell-specific phenomenon and requires the cytoplasmic domain

Class I major histocompatibility (MHC) antigens are expressed by virtually all mammalian cells, yet their levels of expression and behavior on the cell surface vary in a cell-specific fashion. A panel of lymphoid (both B and T) and nonlymphoid cell lines was used to study the kinetics of internalization of the H-2Ld class I MHC in different cell types. These studies revealed that endocytosis of H-2Ld occurs by both constitutive and PMA-regulated pathways in lymphoid cells, but only by a PMA-refractory pathway in the nonlymphoid cells tested. Transfectant derivatives of the T lymphoma, EL4, which express wild-type or mutant H-2Ld class I MHC antigens, were used to investigate the requirement for the cytoplasmic domain of the class I MHC antigen for its endocytosis in T lymphocytes. These studies showed that modification or deletion of the cytoplasmic domain of H-2Ld abrogates endocytosis via a PMA-regulated pathway. The role of cytoplasmic domain phosphorylation in PMA-inducible endocytosis was examined. The wild-type H-2Ld antigen is phosphorylated in all cell types examined, and this phosphorylation is up-regulated by PMA treatment. In contrast, cytoplasmic domain mutants of H-2Ld fail to be phosphorylated in vivo, in the presence or absence of PMA. The universality of PMA-inducible hyperphosphorylation of the class I MHC antigen among diverse cell types leads us to conclude that phosphorylation of the cytoplasmic domain, while perhaps necessary, is not sufficient for triggering endocytosis via a PMA-inducible pathway. Furthermore, the results with the cytoplasmic domain mutants of H-2Ld suggest that a structural conformation of the class I MHC cytoplasmic domain is required for endocytosis via this route.     

Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.     

Cytotoxic T lymphocytes from mice with soluble class I Q10 molecules in their serum are not tolerant to membrane-bound Q10

Q10 is a class I Qa-2 region-encoded molecule that is secreted by the liver and present in serum at high concentrations (about 10 to 60 micrograms/ml) in most strains of mice. The amino terminal portion of this molecule can also be expressed as an integral membrane protein by splicing the 5' end of the Q10 gene to the 3' end of H-2Ld and transfecting the hybrid gene into murine L cells. Because CTL primarily recognize polymorphic determinants controlled by the alpha 1 and alpha 2 domains of class I molecules and because the Q10d/Ld product expressed by transfected L cells includes the alpha 1 and alpha 2 domains of Q10d, we could address whether mice bearing serum Q10 were tolerant to this molecule at the CTL level. The results of these experiments demonstrate that Q10+ mice are able to generate H-2-unrestricted CTL activity against Q10d expressed on transfected L cells, and this response was not inhibitable by the addition of Q10-containing normal mouse serum. It is unlikely that this CTL activity is due to possible polymorphic differences in Q10 alleles, since semisyngeneic BALB/c (H-2d) mice, from which the Q10d hybrid gene construct was derived, are able to generate anti-Q10d effector cells. The Q10d molecule was shown to cross-react with H-2Ld, lending support to the concept that Qa genes can serve as donors for polymorphic sequences found in H-2K, -D, and -L. That mice can generate anti-Q10 CTL activity suggests that this soluble class I protein does not act as a toleragen for these cells. The implications of these findings for an understanding of self-tolerance are discussed.     

Structural and functional dissection of an MHC class I antigen-binding adenovirus glycoprotein.

The early transmembrane glycoprotein E19 of adenovirus-2 binds to class I antigens of the major histocompatibility complex (MHC). The association is initiated in the endoplasmic reticulum of infected cells and abrogates the intracellular transport of the class I molecules. To examine which parts of the E19 molecule are responsible for the association with the class I antigens and which parts confine the protein to the endoplasmic reticulum we have constructed a series of mutated E19 genes, which have been expressed in an improved mammalian expression vector. By various manipulations the membrane anchoring and the cytoplasmic domains were removed from the protein. The biosynthesis of the mutant protein was examined. All mutant proteins were secreted from the cells suggesting that the transmembrane and/or cytoplasmic portions of the E19 molecule are responsible for its confinement to the endoplasmic reticulum. The ability to associate with class I antigens was retained by the lumenal domain of the E19 protein.     

The alpha-3 domain of class I MHC proteins influences cytotoxic T lymphocyte recognition of antigenic determinants located within the alpha-1 and alpha-2 domains

An interspecies class I MHC molecule, Kb1+2/A2 (in which the alpha-1 and alpha-2 domains of the H-2Kb molecule have been linked to the alpha-3, transmembrane and intracytoplasmic domains of the HLA-A2 molecule) has been expressed on both human and mouse target cells by gene transfer. Maintenance of serologic determinants has been demonstrated. However, decreased lysis by allospecific CTL populations of cell lines that expressed a hybrid interspecies class I molecule, Kb1+2/A2, as compared with lines that expressed the native Ag, H-2Kb, has been described. An analysis with a limited panel of H-2Kb allospecific clones demonstrated that not all H-2Kb-specific CTL can lyse cells that express Kb1+2/A2 Ag. This suggested that the reduction of lysis by CTL populations was due to the loss of specific alloreactive clones in the population. Each clone used in this study was then defined as having high or low affinity characteristics. No correlation between the affinity of the CTL and the ability to recognize the interspecies hybrid molecule could be shown. Rather, these data suggest that antigenic determinants that are located within the polymorphic domains, alpha-1 and alpha-2, may be conformationally influenced by the alpha-3 domain.     

Expression and function of transplantation antigens with altered or deleted cytoplasmic domains

Two mutants of the class I gene encoding the H-2L^d transplantation antigen have been constructed. In one mutant the cytoplasmic domain of the class I molecule has been altered by deletion of 24 of the 31 C-terminal residues, and in the second the C-terminal 25 residues of the cytoplasmic domain have been replaced with a unique sequence of 19 amino acids. These mutant class I genes have been transferred into mouse L cells by DNA-mediated gene transfer. Both mutant genes are expressed at normal levels on the cell surface, and they have charge properties and sizes consistent with the introduced alterations. These mutant L^d molecules can serve as target antigens for allogeneic cytotoxic T cells and as restricting elements for virus-specific cytotoxic T cells. These results show that the 24 residues replaced or deleted from the carboxy terminus of the class I molecule are not required for its transport to or integration in the plasma membrane, nor for its function as a target antigen or a restricting element during T-cell-mediated cytotoxicity.     

A single gene encodes soluble and membrane-bound forms of the major histocompatibility Qa-2 antigen: anchoring of the product by a phospholipid tail

The H-2, Qa, and Tla genes of the murine major histocompatibility complex are related to each other by DNA sequence homology. The H-2 genes encode ubiquitously expressed transplantation antigens that serve as recognition structures for cytotoxic T cells. The identities of the Qa and Tla products, their sites of expression, and their functions are largely unknown. We report here that the Qa region gene Q7 encodes a membrane-bound as well as a secreted form of the serologically defined antigen Qa-2. The Q7 gene introduced into liver-derived cells is expressed as a membrane-bound and as a secreted molecule. In transfected L cells it is expressed only as a soluble protein. Biochemical analysis suggests that the Q7 product is anchored to the liver cell membranes by a phospholipid tail. This feature may be responsible for cell type-specific expression of the two forms of the Qa-2 molecules.     

CTL and serologically defined antigens of B2m,H-3 region

The antigens of the B2m,H-3 region of 13 chromosome 2 congenic strains and seven inbred strains have been studied by using CML and serologic techniques. Nine patterns of cross-reactivity have been defined by CML assays. These results are in agreement with an extend previously described cross-reactivity studies. The reactivities of three monoclonal antibodies previously thought to be reacting with B2M-B are shown to differ: Ly-m11 and J-5 react with cells of strain B10-pa,at and clone 23 does not. Two H-3 region loci are hypothesized on the basis of CML and serologic activity: B2m and H-3. The CTL responses to the B2M antigens are H-2K restricted; the CTL responses to H-3 antigens are H-2D restricted. The restriction of the response to the H-3 antigen requires effector-target identity of the H-2D molecule but not the B2M molecule of the class I antigen. These loci have been separated by recombination from H-42 in the production of the congenic strain B10.FS-a. A gene order of B2m, H-3, H-42 is suggested.     

The nucleotide sequence and comparative analysis of the H-2Dp class I H-2 gene

We present the complete nucleotide sequence and the deduced amino acid sequence of the H-2Dp class I gene. This gene, which was cloned from a B10.P genomic DNA library, encodes and intact, functional H-2Dp molecule. Comparative analysis of the Dp sequence with other class I sequences reveals both similarities and differences. This analysis also shows that these genes exhibit D region-specific, locus-specific, as well as allele-specific sequences. The H-2Dp nucleotide sequence is greater than 90% homologous to the H-2Ld and H-2Db genes and only approximately 85% homologous to the H-2Dd gene. The K region and Qa region genes are less homologous. The 3' noncoding sequences appear to be region-specific. All of the previously described D region genes, Db, Ld, and Dd, possess the B2-SINE Alu-like repetitive sequence, as does Dp. Thus, this B2 repeat is a region-specific marker present in all D region genes studied so far. The additional polyadenylation site found in the H-2Dp gene starting at nucleotide 4671, which is homologous to non-D region sequences, as well as unique protein Dp coding sequences, make this gene an interesting model for studying the evolution of polymorphism and structure/function relationships in the class I gene family.     

Expression of class I genes in the major histocompatibility complex: identification of eight distinct mRNAs in DBA/2 mouse liver

The mouse H-2 multigene family includes the genes coding for the major transplantation antigens and for genes located in the Qa-TIa region. We have studied a collection of class I cDNA clones made from liver mRNA of DBA/2 mice (H-2d haplotype) and found that at least six distinct class I genes are transcribed, including three genes of the Qa-TIa region. Two of these six genes each yield two distinct mRNAs, resulting from alternate splicing. Altogether, liver cells may express at least eight distinct class I polypeptides, of which three might be secreted, while one may be a new presumptive nonpolymorphic surface antigen.